Angiogenesis is the development of a blood supply to a given area of tissue. Angiogenesis is part of normal embryonic development and revascularization of wound beds, as well as due to the stimulation of vessel growth by inflammatory or malignant cells. Angiogenesis is also the process through which tumors or inflammatory conditions derive a blood supply through the generation of microvessels.
Angiogenesis is regulated in normal and malignant cancer tissues by the balance of angiogenic stimuli and angiogenic inhibitors that are produced in the target tissue and at distant sites (See, Fidler et al., [1998]; and McNamara et al., [1998]). Vascular endothelial growth factor-A (VEGF, also known as vascular permeability factor, “VPF”) is a primary stimulant of angiogenesis. VEGF is a multifunctional cytokine that is induced by hypoxia and oncogenic mutations and can be produced by a wide variety of tissues (See, Kerbel et al., [1998]; and Mazure et al., [1996]).
The recognition of VEGF as a primary stimulus of angiogenesis in pathological conditions has led to various attempts to block VEGF activity. Inhibitory anti-VEGF receptor antibodies, soluble receptor constructs, antisense strategies, RNA aptamers against VEGF and low molecular weight VEGF receptor tyrosine kinase (RTK) inhibitors have all been proposed for use in interfering with VEGF signaling (See, Siemeister et al., [1998]). In fact, monoclonal antibodies against VEGF have been shown to inhibit human tumor xenograft growth and ascites formation in mice (See, Kim et al., [1993]; Asano et al., [1998]; Mesiano et al., [1998]; Luo et al., [1998a] and [1998b]; and Borgstrom et al., [1996] and [1998]).
RTKs comprise a large family of transmembrane receptors for polypeptide growth factors with diverse biological activities. The intrinsic function of RTKs is activated upon ligand binding, which results in phosphorylation of the receptor and multiple cellular substrates, and subsequently in a variety of cellular responses. (See, Ullrich & Schlessinger, Cell 61:203-212 [1990]).
Angiogenesis, involving VEGF and RTKs is not only involved in cancer development, as many other diseases or conditions affecting different physiological systems are angiogenesis-dependent, such as arthritis and atherosclerotic plaques (bone and ligaments), diabetic retinopathy, neovascular glaucoma, macular degeneration, ocular herpes, trachoma and corneal graft neovascularization (eye), psoriasis, scleroderma, rosacea, hemangioma and hypertrophic scarring (skin), vascular adhesions and angiofibroma (blood system).
VEGF is an angiogenesis factor of major importance for skin vascularization (Detmar [2000]). VEGF expression is upregulated in the hyperplastic epidermis of psoriasis (Detmar and Yeo et al. [1995]), in healing wounds and in other skin diseases characterized by enhanced angiogenesis (Detmar [2000], supra). Targeted overexpression of VEGF in the epidermis of transgenic mice was reported to result in enhanced skin vascularization with equal numbers of tortuous and leaky blood vessels (See e.g., Brown et al., [1998]). Also, chronic synthesis of VEGF in mouse skin leads to the first histologically equivalent murine model of human psoriasis (Xia et al., [2003]) that is reversible by binding agents specific for VEGF.
The Bowman-Birk protease inhibitor (BBI) is a designation of a family of stable, low molecular weight trypsin and chymotrypsin enzyme inhibitors found in soybeans and various other seeds, mainly leguminous seeds and vegetable materials. BBI comprises a family of disulfide bonded proteins with a molecular weight of about 8 kD (See e.g., Chou et al., Proc. Natl. Acad. Sci. USA 71:1748-1752 [1974]; Yavelow et al., Proc. Natl. Acad. Sci. USA 82:5395-5399 [1985]; and Yavelow et al., Cancer Res. (Suppl.) 43:2454s-2459s [1983]). BBI has a pseudo-symmetrical structure of two tricyclic domains each containing an independent native binding loop, the native loops containing binding sites for both trypsin and chymotrypsin (See, Liener, in Summerfield and Bunting (eds), Advances in Legume Science, Royal Bot. Gardens, Kew, England). These binding sites each have a canonical loop structure, which is a motif found in a variety of serine proteinase inhibitors (Bode and Huber, Eur. J. Biochem., 204:433-451 [1992]). Commonly, as in one of the soybean inhibitors, one of the native loops inhibits trypsin and the other inhibits chymotrypsin (See, Chen et al., J. Biol. Chem., 267:1990-1994 [1992]; Werner & Wemmer, Biochem., 31:999-1010 [1992]; Lin et al., Eur. J. Biochem., 212:549-555 [1993]; and Voss et al., Eur. J. Biochem., 242:122-131 [1996]) though in other organisms (e.g., Arabidopsis), both loops are specific for trypsin.
STI inhibits the proteolytic activity of trypsin by the formation of a stable stoichiometric complex (See e.g., Liu, Chemistry and Nutritional Value of Soybean Components, In: Soybeans, Chemistry, Technology and Utilization, pp. 32-35, Aspen Publishers, Inc., Gaithersburg, Md., [1999]). STI consists of 181 amino acid residues with two disulfide bridges and is roughly spherically shaped (See e.g., Song et al., J. Mol. Biol., 275:347-63 [1998]). The trypsin inhibitory loop lies within the first disulfide bridge. The Kunitz-type soybean trypsin inhibitor (STI) has played a key role in the early study of proteinases, having been used as the main substrate in the biochemical and kinetic work that led to the definition of the standard mechanism of action of proteinase inhibitors.
Eglin C is a small monomeric protein that belongs to the potato chymotrypsin inhibitor family of serine protease inhibitors. The proteins that belong to this family are usually small (60-90 amino acid residues in length) and contain no disulfide bonds. Eglin C, however, is highly resistant to denaturation by acidification or heat regardless of the lack of disulfide bonds to help stabilize its tertiary structure. The protein occurs naturally in the leech Hirudo medicinalis. 